Role of host & bacterial factors in persistence of Salmonella in the bovine lymphatic system

Lead Research Organisation: University of Edinburgh
Department Name: The Roslin Institute

Abstract

Salmonella is a bacterium that causes severe diarrhoea in humans. Infections are often acquired via the food chain and environment from farmed animals owing to the ability of Salmonella to persist in their bodies. Around 94 million human cases and 155 thousand deaths occur worldwide each year. Cattle are a significant source of such infections, partly because Salmonella can leave the bovine gut via a branching network of vessels and nodular glands termed the lymphatic system. The lymphatic system normally helps to fight infection, however some types of Salmonella are adapted to survive in lymph nodes and evade the immune system. Removal of such lymph nodes is not possible on the scale of modern beef production as they are small, widely dispersed and hard to access. As a consequence, they are often incorporated into ground beef for human consumption. Large outbreaks due to ground beef have occurred, including by strains resistant to frontline medicines, and a need exists to devise strategies to prevent or reduce Salmonella infection in cattle. Such strategies are also required to enhance animal health as Salmonella are a significant cause of diarrhoea, blood-poisoning and abortion in cattle.

The Stevens laboratory has been at the forefront of research to understand how Salmonella colonises the intestines of cattle and, in some cases, migrates around the body via the lymphatic system. We have developed a novel surgical model in which we insert tubes into the blood and lymphatic systems to capture bacteria as they migrate from the gut. This has shown that escape from lymph nodes via the lymphatic system can partly explain why some strains spread around the body, whereas others are confined to the gut or cleared. A key gap in our knowledge is how Salmonella enters the lymphatic system in the first place. Almost nothing is known about the host cell types with which Salmonella associates in the bovine gut or whether these migrate to the lymphatic system. The consequences of such interactions for Salmonella and the development of protective immune responses are unknown. Such gaps in knowledge are constraining our ability to design strategies to control infection. The Hopkins and Hope laboratories conduct world-leading research to understand the nature and consequences of interactions between infectious agents and cells of the ruminant immune system, which in turn is helping to improve vaccines for tuberculosis, Johne's disease, viruses and parasitic infections. Our proposal brings together substantial cash investment from Pfizer Animal Health and researchers with complementary expertise to:

1. Understand how Salmonella enters the lymphatic system of cattle. This will involve inserting tubes into lymph vessels draining to lymph nodes at various sites in the gut of young calves and older steers. We will examine which cell types interact with Salmonella, how they respond and what happens to the bacteria. We will then ask whether such events can explain why some types of Salmonella are cleared by cattle whereas others spread around the body. The information will help us to target vaccines to relevant cell types and boost beneficial responses.

2. Determine if different cattle-associated strains of Salmonella are equally able to spread through the lymphatic system. This will tell us whether vaccines or drugs must target a wide range of Salmonella strains, or focus on high-risk types.

3. Identify Salmonella genes required for survival in the lymphatic system. We recently used a novel method to simultaneously survey the ability of hundreds of Salmonella mutants, each lacking a different character, to colonise the bovine gut wall. We are able to retrospectively apply this method to lymph nodes from the same animals to identify factors that influence persistence in the lymphatic system. Such factors may be suitable for inclusion into vaccines or as targets for drugs.

Technical Summary

Persistence of Salmonella in the bovine lymphatic system leads to contamination of ground beef and is associated with the systemic virulence of host-restricted serovars. The mode of delivery of Salmonella from the intestinal mucosa to draining lymph nodes and the nature and consequences of interactions with host cells in this process are unknown. Moreover, it is unclear if cattle-associated serovars are equally able to enter the bovine lymphatic system and evade clearance. This proposal brings together complementary expertise and substantial investment from Pfizer Animal Health to define the role of host and bacterial factors in persistence of Salmonella in the bovine lymphatic system. Specifically, we will define the mode of translocation of S. Dublin to lymph nodes draining the intestines of calves and steers by cannulation of afferent lymph vessels. We will define the cell tropism of S. Dublin, fate of the intracellular bacteria and cellular responses, and determine how such events vary with serovar Typhimurium, Gallinarum and Newport strains toward an explanation of their differential virulence in cattle. We will also examine the spatio-temporal dynamics of spread of signature-tagged strains of cattle-associated serovars to define the extent to which interventions must confer cross-serovar protection. We also propose to identify Salmonella genes mediating persistence in the bovine lymphatic system by retrospective application of transposon-directed insertion-site sequencing to archived lymph nodes from cattle infected with 8550 random S. Typhimurium mutants. We have assigned phenotypes for >90% of such mutants in the distal ileal mucosa and quantitative assessment of their abundance in cognate nodes will identify niche-specific virulence factors and targets for intervention. The project extends our productive BBSRC-funded research using unique models in a species of strategic importance to food security and safety.

Planned Impact

Cattle play an important role in global food security and bovine salmonellosis constrains both animal welfare and food safety. Contamination of ground beef is a significant concern, particularly in the United States where large multi-state outbreaks have occurred. Salmonella evolution has been punctuated by the emergence of epidemic and multi-drug resistant variants that have caused substantial harm in cattle and humans (e.g. S. Typhimurium definitive-types 104, 204 and 204c). The basis of the rise and decline of epidemic variants and reasons why some serovars cause acute diarrhoea while others cause sepsis and abortion are not understood. Prophylactic use of antibiotics is prohibited in many countries and therapeutic use is restricted owing to the potential for entry of residues into the food chain. Removal of contaminated lymphatic tissue is not feasible on the scale of modern beef production, where some abattoirs in the US process >5000 cattle a day. In this context, vaccines that prevent persistence of Salmonella in the bovine lymphatic system, or treatments that accelerate clearance, are highly desirable. The project will yield valuable data to underpin the design of such strategies, as reflected in the substantial investment of Pfizer Animal Health - a global leader in the development of veterinary vaccines and medicines. The project will also yield novel data on the repertoire and function of antigen-presenting cells serving the bovine intestines, and the mechanisms leading to protective innate and adaptive responses to enteric pathogens. Such data will benefit academia and industry in their efforts to control other ruminant intestinal diseases, including those caused by viruses and parasites. It may also identify immune signatures associated with infection or vaccination that can be exploited in diagnostic tests, as evidenced by an award-winning assay devised by Dr Hope to differentiate BCG-vaccinated cattle from those with tuberculosis. Identification of serovars and genes associated with persistence of Salmonella in the bovine lymphatic system will also yield markers for risk analysis and aid the targeting of control strategies.

Intellectual property arising from the project will be identified, protected and exploited as described in Pathways to Impact. Though we will primarily focus our impact activities on control of zoonosis and cattle health via links with Pfizer, the project will also benefit those studying other host-pathogen interactions and instil training in diverse areas. In particular, it will nurture skills in molecular microbiology, immunology, pathogenesis and surgery in animals of significance to food supply and safety. Training will extend beyond those deployed on the project to undergraduate, Masters and doctoral students via the research-led teaching of the applicants. Academics and Policy Makers will be informed of the nature and implications of the research via scientific and lay publications, presentations at symposia, invited lectures and via the comprehensive websites of the investigators. Moreover, the applicants actively engage with varied societies and forums to discuss issues affecting animal health and strategies for disease control. Materials and data arising from the project will be made available for legitimate uses on request, subject to publication and scrutiny for IP. Exchange of staff and students will promote knowledge transfer.

The project will also raise issues and data of importance to the public. The applicants have used short films, public speeches and lay articles to convey the purpose and importance of their work. This has included public engagement on food safety, ruminant diseases and methods to reduce, refine and replace animal use in research. The investigators will also explore ways to educate school children, for example by extending established links with the National Centre for Biotechnology Education and the STEM ambassador scheme.

Publications

10 25 50
 
Description The project focuses on the role of bacterial and host factors on the entry and persistence of Salmonella in the bovine lymphatic system. In particular we have defined the nature and consequences of interactions between Salmonella serovars and bovine antigen-presenting cells, both in the intestines and associated lymph nodes of cattle and using cells cultured ex vivo. We have retrospectively applied transposon-directed insertion-site sequencing to a comprehensive library of S. Typhimurium mutants screened in calves, using output pools from archived mesenteric lymph nodes and proximal ileal mucosa. This has assigned phenotypes to thousands of S. Typhimurium genes during colonisation of the gut and draining mesenteric lymph nodes and identified a subset of candidate genes that appear to play a specific role in entry or persistence of Salmonella in lymph nodes that are not required in the intestinal mucosa. Validation of mutant phenotypes is ongoing at the time of writing. We have also devised novel methods to track the fate of serovars in mixed pools as they spread through inoculated cattle by massively-parallel sequencing of polymorphic alleles (see research tools). This is allowing us to assess the risk posed by different Salmonella serovars to food safety and animal health. As a 3R method, we believe it will find wider applications in the study of bacterial infections. Three manuscripts are in preparation.
Exploitation Route Our findings inform the design of strategies to control Salmonella in cattle, notably by defining the role of thousands of S. Typhimurium genes in colonisation of the gut and lymphatic system. Our use of a novel method to follow the fate of serovars in mixed infections has also indicated that vaccines for control of Salmonella in cattle are likely to be required to be cross-protective for multiple serovars, as most of those tested in mixed infections were able to enter and persist in the lymphatic system. This also informs those conducting risk analysis in meat processing, as the content of one lymph node could not reliably predict the content of the next and consideration of our data will be required when devising sampling strategies to assess food safety.
Sectors Agriculture, Food and Drink,Pharmaceuticals and Medical Biotechnology

 
Description BBSRC Industrial Partnering Award
Amount £568,230 (GBP)
Funding ID BB/K015524/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom of Great Britain & Northern Ireland (UK)
Start 11/2013 
End 03/2017
 
Title Strategy for quantifying individual bacterial strains during mixed infections 
Description We devised a novel strategy to follow the fate of individual bacterial strains during mixed infections. Specifically, we wished to follow the fate of multiple Salmonella enterica serovars during colonisation of the bovine host. These are indistinguishable by culture, and while they can be specifically detected by serology, quantification of numbers of each serovar during mixed infection would involve analysis of many hundreds or thousands of individual colonies. We devised a method based on massively-parallel sequencing of a polymorphic allele (rpoB), whereby sequence reads can be used to identify the strain present (based on single nucleotide polymorphisms specific to each strain) and the number of sequence reads can be taken as a measure of the abundance of the cognate strain. Using this method we were able to simultaneously define the fate of 12 different S. enterica strains during infection of cattle, including as they colonised the gut over time and spread from the gut to the lymphatic system and other tissues. This 3R approach should allow phenotypes to be derived with reduced use of animals in experiments and could, for example, be used to see if vaccine-induced responses are effective in control of all the different members of a bacterial population present - in this case toward a pan-serovar cross-protective vaccine. A manuscript reporting the tool and our findings is in preparation at the time of writing. 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact Too early as only validated toward the end of 2016. 
 
Description Collaboration with Kansas State University 
Organisation Kansas State University
Country United States of America 
Sector Academic/University 
PI Contribution Kansas State University are partners in this BBSRC US-UK Partnering Award related to Salmonella and E. coli infections in food-producing animals.
Collaborator Contribution Sharing of expertise & materials.
Impact None yet from this specific partner.
Start Year 2014
 
Description Collaboration with Texas Tech University 
Organisation Texas Tech University
Country United States of America 
Sector Academic/University 
PI Contribution Partner in this BBSRC US-UK Partnering Award related to Salmonella and E. coli infections in food-producing animals.
Collaborator Contribution We hosted a visiting postdoctoral research fellow (Marie Bugarel from TTU) in 2015 and provided training in methods to study the basis of Salmonella virulence (inc. mutagenesis, cell-based assays & in vivo models).
Impact A manuscript relating to a novel method to follow the fate of Salmonella enterica serovars during mixed infections is in preparation at the time of writing. This arose, in part, from the collaboration funded by this partnering award (see tools), with resources from another BBSRC- and Zoetis-funded project (BB/K015524/1).
Start Year 2014
 
Description Collaboration with United States Department of Agriculture 
Organisation U.S. Department of Agriculture USDA
Country United States of America 
Sector Public 
PI Contribution USDA are partners in this BBSRC US-UK Partnering Award related to Salmonella and E. coli infections in food-producing animals. We have shared expertise, ideas & materials. A co-Investigator at the Roslin Institute (Professor John Hopkins) visited USDA researchers to transfer his expertise in surgical cannulation of lymphatic vessels, to allow the team to gain access to Salmonella and immune cells as they migrate from the intestines of cattle.
Collaborator Contribution Shared expertise, ideas & materials.
Impact Not at this stage.
Start Year 2014
 
Description Collaboration with Zoetis 
Organisation Zoetis
Country United States of America 
Sector Private 
PI Contribution The project was a BBSRC Industrial Partnership Award with Zoetis (formerly Pfizer Animal Health) to understand the role of bacterial and host factors in colonisation of the bovine lymphatic system by Salmonella. We analysed the nature and consequences of Salmonella interactions with immune cells in the gut and lymphatic system of cattle, assigned phenotypes to thousands of Salmonella genes during lymph node colonisation and analysed the relative ability of different Salmonella serotypes to enter and persist in the lymphatic system using a novel strain tracking method. The data aid the design of strategies to control Salmonella in cattle, both to benefit bovine health and reduce the impact of zoonotic infections.
Collaborator Contribution Zoetis provided $250 toward project costs and also hosted meetings of a Salmonella Research Cluster that also involved academics at Texas Tech University, Kansas State University and the US Department of Agriculture. In turn this helped to form the basis of a BBSRC US-UK Partnering Award (BB/L026732/1).
Impact See key findings.
Start Year 2012